Spotted seals (Phoca largha) harbor unique gut microbiota shaped by their host habitat.

Assessing the structure and composition of gut microbiota of sentinel species such as spotted seals (Phoca largha) is a potential tool for assessing the health of the marine mammals and their habitats. However, the link between the host microbiome and their habitat is poorly understood. In this study, microbial communities in the habitat (sea ice and water) and marine mammalian host (fecal matter from P. largha) were evaluated in samples obtained from the Liaodong Bay, China during population aggregation period. Results from high-throughput sequencing showed that the bacterial communities in P. largha fecal matter were less rich and diverse compared to those from the water and ice samples. Significant differences in the composition and function of bacterial communities were also found among the water, ice, and fecal samples, in which sample type and sampling site had the greatest impact on composition and function variations, respectively. Several potential pathogenic bacteria and bacteria with functions associated with human disease were significantly enhanced in the communities of P. largha feces compared to those of surrounding environments. The ratios of environmental microorganisms sourced from the P. largha fecal matter were estimated. The results showed that certain bacteria in P. largha-inhabited fecal matter were associated with sea ice and had specific antibiotic resistance and infectious capacity. These findings provide critical data for monitoring the health of marine mammals and their habitats, which is essential for predicting the impact of anthropogenic disturbances on marine ecosystems.

Integrated proteomics and metabolomics reveal the variations in the physiological state of spotted seal (Phoca largha) pups following artificial rescue.

Spotted seals (Phoca largha) are a critically endangered pinniped in China. Artificial rescue of newborn pups is a conventional method to enhance their survival and maintain the population. However, little is known about the variations in the physiological state of spotted seal pups following artificial rescue. Here, an integrated proteomics and metabolomics study was performed on spotted seal pups by using whole blood samples to characterize the molecular response to artificial rescue. The proteome was characterized as having 1165 proteins that were predominantly associated with the metabolic pathways, and the complement and coagulation cascades. Remarkable variation was found in spotted seal pup blood following artificial rescue, whereby the levels of 193 proteins and 32 metabolites significantly varied in some metabolic pathways, including glycosylphosphatidylinositol-anchor biosynthesis, focal adhesion, cardiac muscle contraction, and fatty acid beta-oxidation. After rescue, spotted seal pups had a higher risk to mild hemolytic disorder due to the energy metabolism in the red blood cells was possibly suppressed. Moreover, spotted seal pups after rescue could have stronger anaerobic exercise abilities, while their capacity for long-term high-intensity exercise was weaker.

Spatial distribution and ecological risks of polycyclic aromatic hydrocarbons in sea ice and seawater from northern Liaodong Bay, China.

To better understand the spatial distribution and ecological risks of polycyclic aromatic hydrocarbons especially in low latitude coastal productive areas, PAHs in sea ice were examined for the first time in northern Liaodong bay of China in December 2020. Results showed ΣPAHs were dominated by 2- and 3-ring, with the mean concentration of 241.9 ng L-1 and 202.8 ng L-1 in sea ice and seawater, respectively, suggesting a moderate ecological risk based on Risk Quotients assessment. Ice enrichment factors were greater than 1 at 82% of the sampling sites, reflecting enrichment of PAHs in sea ice. The characteristic compounds ratios demonstrated PAHs mainly derived from petrogenic sources, while combustion was another crucial source for PAHs in sea ice via atmospheric deposition. This helps to better elucidate pollution status, potential sources and risk assessment of PAHs in productive coastal oceans especially during ice-covered period for contamination control and environmental management.

Tissue distribution and health risk of trace elements in East Asian finless porpoises.

We investigated the tissue distribution, trophic transfer, and ecological risk of 13 trace elements in 26 East Asian finless porpoises (Neophocaena asiaeorientalis sunameri), an endangered species found in the Liaodong Bay and the north Yellow Sea. All the investigated trace elements were detected in the tissue and food web of the East Asian finless porpoises. The concentrations of the potentially toxic elements were 2.37 × 10-5 - 754 mg kg-1 dry weight (dw) in stranded porpoises and 0.01-159 mg kg-1 dw in their food web. Tissue-specific distribution of the trace elements generally ranked as: liver > kidney > heart > lung > muscle. Zn was the dominant contaminant in the five investigated tissues. Significant positive correlations were found between body length or age and some trace elements, especially Cd. Adults (≥2 years old) presented higher concentrations of most of the trace elements than juveniles (<2 years old). Sex-dependent distribution of the trace elements was insignificant except for Mn, Ni, and Zn in muscle and renal tissue. As, Cu, Mn, Ni, Pb, and V biodiluted across the East Asian finless porpoise food web while Zn biomagnified. However, Hg, Cd, Co, Cr, Se, and Sn did not exhibit apparent trophic transfer trends. Overall, ecological risk assessment of trace elements in East Asian finless porpoises suggested that greater attention should be given to Hg, As, Cd, and Se.

Age-associated variation in the gut microbiota of chinstrap penguins (Pygoscelis antarctica) reveals differences in food metabolism.

Age is known to affect the gut microbiota in various animals; however, this relationship is poorly understood in seabirds. We investigated the temporal succession of gut microbiota in captive chinstrap penguins of different ages using high-throughput sequencing. The gut microbiota exhibited a significant age succession pattern, reaching maturity in adults and then declining with increasing age. Only 15 amplicon sequence variants were shared among the gut microbiota in chinstrap penguins at all studied ages, and these contributed to most of the age-related variations in total gut microbiota. Co-occurrence networks found that these key bacteria belonged to the genera Acinetobacter, Clostridium sensu stricto, and Fusobacterium, and more species interactions were found within the same taxonomy. Functional prediction indicated that most of the metabolic functions were more abundant in the gut microbiota in adult chinstrap penguins, except for carbohydrate metabolism, which was significantly more abundant in older individuals.

Proteomics reveals the preliminary physiological states of the spotted seal (Phoca largha) pups.

Spotted seal (Phoca largha) is a critically endangered pinniped in China and South Korea. The conventional method to protect and maintain the P. largha population is to keep them captive in artificially controlled environments. However, little is known about the physiological differences between wild and captive P. largha. To generate a preliminary protein expression profile for P. largha, whole blood from wild and captive pups were subjected to a label-free comparative proteomic analysis. According to the results, 972 proteins were identified and predicted to perform functions related to various metabolic, immune, and cellular processes. Among the identified proteins, the expression level of 51 were significantly different between wild and captive P. large pups. These differentially expressed proteins were enriched in a wide range of cellular functions, including cytoskeleton, phagocytosis, proteolysis, the regulation of gene expression, and carbohydrate metabolism. The abundances of proteins involved in phagocytosis and ubiquitin-mediated proteolysis were significantly higher in the whole blood of wild P. largha pups than in captive individuals. In addition, heat shock protein 90-beta, were determined as the key protein associated with the differences in the wild and captive P. largha pups due to the most interactions of it with various differentially expressed proteins. Moreover, wild P. largha pups could be more nutritionally stressed and have more powerful immune capacities than captive pups. This study provides the first data on the protein composition of P. largha and provides useful information on the physiological characteristics for research in this species.

Distribution of microbiota across different intestinal tract segments of a stranded dwarf minke whale, Balaenoptera acutorostrata.

Marine mammals are an important part of ocean ecosystems, of which, whales play a vital role in the marine food chain. In this study, the mucosa and contents from different intestinal tract segments (ITSs) of a stranded dwarf minke whale (Balaenoptera acutorostrata) were analyzed. The gut microbiota were sequenced using high-throughput sequencing technology, based on a 16S rRNA approach. The microbial composition of the intestinal mucosa and its contents were similar in every single ITS. Large intestine microbiota richness and diversity were significantly higher when compared to the duodenum and jejunum. The dominant bacteria in the gut were Firmicutes and Actinobacteria; the former was enriched in the large intestine, whereas the latter was more abundant in the duodenum and jejunum. Our findings provide novel insights for microbiota in B. acutorostrata.

Identification of the complete mitochondrial genome of the king penguin Aptenodytes patagonicus (Sphenisciformes: Spheniscidae: Aptenodytes).

The complete mitochondrial genome of the king penguin Aptenodytes patagonicus was firstly determined. The mitogenome is 17,477 bp in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. The total nucleotide composition is 31.0% A, 22.2% T, 33.1% C, and 13.8% G, with a total A + T content of 53.2%. The phylogenetic analysis demonstrates a close relationship between A. patagonicus and A. forsteri. These results provide fundamental information for further phylogeny and genetic studies on Aptenodytes genus.

Classification of underwater vocalizations of wild spotted seals (Phoca largha) in Liaodong Bay, China.

Underwater vocalizations were recorded and classified from wild spotted seals (Phoca largha) in Liaodong Bay, China. The spotted seals exhibited an extensive underwater vocal repertoire but with limited complexity. Four major call types, representing 77.8% of all calls recorded, were identified using multivariate analyses of ten acoustic parameters; knock, growl, drum, and sweep. The calls were relatively brief (12-270 ms, mean of -10 dB duration) pulsating sounds of low-frequency (peak frequency <600 Hz) and narrow bandwidth (169-232 Hz, mean of -3 dB bandwidth; 237-435 Hz, mean of -6 dB bandwidth). Frequency variables (-3/-6 dB frequency bandwidth, center frequency, and top three peak frequencies) were the primary descriptors used to differentiate the call types. Comparing the spotted seal underwater vocalizations with those of the closely related Pacific harbor seal (Phoca vitulina richardii) indicated that the two species use similar bandwidths and peak frequencies but spotted seal calls were generally shorter. Knowledge of underwater vocalizations of wild spotted seals is important for understanding the species behavior and for planning future acoustic surveys of its distribution and occurrence.

In-air vocal repertoires of spotted seals, Phoca largha.

Spotted seals (Phoca largha) are thought to be less vocal than other phocids. However, acoustic communication behaviors of spotted seals have been reported several times. In this study, the vocal repertoires of spotted seals housed in Dalian Sun Aquarium, China were recorded and analyzed. The frequencies of the sounds made by the seals ranged from 139.3 to 2323.1 Hz, and the time durations lasted from 92.8 to 1208 ms, depending on age and gender (P < 0.01). The peak-to-peak sound source levels were 109-124 dB re 20µPa. In total, seven vocal types were identified: pup call, yearling call, bark, growl, grunt, moo, and throat guttural. The pups emitted sounds with high frequencies (F1: 972.4 ± 374.4 Hz, mean ± standard deviation) and medial time durations (564 ± 178 ms); when the pups grew older, the sounds became yearling calls, which had high frequencies with median (interquartile range) of 1198.0 (821.7-1385.5) Hz; and long time durations [902 (745-1080) ms]. The male adults emitted sounds with low frequencies [430.2 (388.2-486.7) Hz] and short time durations [334 (233-599) ms], while the female adults emitted sounds with medial frequencies [814.5 (592.6-1024.3) Hz] and medial time durations [531 (336-688) ms].

Serum testosterone, progesterone, and estradiol concentrations and sexual maturation in spotted seals (Phoca largha).

Spotted seals (Phoca largha) are ice-breeding phocid found in eight different breeding colonies all over the world. They exhibit a seasonal breeding pattern, with annual and synchronous cycles; however, little is known about their reproductive endocrinology. In this study, we measured serum testosterone, progesterone, and 17ß-estradiol concentrations in captive spotted seals (simple number: female n = 68; male n = 89) throughout a full reproductive cycle. Males that were older than 4 years had significant testosterone fluctuations and were, therefore, classified as sexually mature. These animals show significant seasonal changes in testosterone levels, with average peak concentrations of 10.81 ± 9.57 nmol/L (±SD) from November to February, compared with mean concentrations of 1.42 ± 3.09 nmol/L throughout the remainder of the year. Females that reported a significant variation in progesterone concentrations and were older than 4 years were considered to be sexually mature. In these females, progesterone levels increased in February, remained elevated for 7 months with a mean value of 37.39 ± 17.03 nmol/L, and then dropped to 0.74 ± 0.54 nmol/L. Serum 17ß-estradiol levels were also found to be significantly increased in January, remained so for 8 months (15.80 ± 14.15 ng/L), and then declined after August (7.77 ± 6.78 ng/L). In seals, mating typically occurs in February and March, 1 month after the observed peaks in testosterone and estradiol concentrations and corresponding to the increase in progesterone. A moderate positive correlation between testosterone and progesterone concentrations in sexually mature males was also observed (Spearman rho, r = 0.63, P < 0.01). In sexually immature females, progesterone and estradiol concentrations were found to be significantly lower than those in mature females. Finally, the observed patterns of estradiol and progesterone in sexually mature females suggest that embryonic diapause or successful implantation occurs in August.

De novo assembly and characterization of spotted seal Phoca largha transcriptome using Illumina paired-end sequencing.

Spotted seal (Phoca largha) is categorized as a critically endangered species in China. The aim of this study was to investigate spotted seal transcriptome by the approach of Illumina paired-end sequencing technology. We obtained a total of 52,146,394 reads for the mixed tissues of liver and spleen from the spotted seal. The de novo assemblies yielded 354,014 contigs and 178,466 unigenes. In the transcriptome, 193 unigenes were assigned to defense mechanisms. Three unigenes encoded MHC class I and 17 unigenes encoded MHC class II. In addition, bioinformatics analysis revealed a total of 4425 simple sequence repeats (SSRs). Fifty SSRs were randomly selected to validate amplification and determine the degree of polymorphism in the genomic DNA pools. Thirty-five primer pairs successfully amplified the expected DNA fragments and detected significant polymorphism among 28 spotted seal individuals. These results would contribute to the understanding of the genetic makeup of spotted seal transcriptome and provide useful information for functional genomic research in this species.

Sex identification of four penguin species using locus-specific PCR.

Traditional methods for sex identification are not applicable to sexually monomorphic species, leading to difficulties in the management of their breeding programs. To identify sex in sexually monomorphic birds, molecular methods have been established. Two established primer pairs (2550F/2718R and p8/p2) amplify the CHD1 gene region from both the Z and W chromosomes. Here, we evaluated the use of these primers for sex identification in four sexually monomorphic penguin species: king penguins (Aptenodytes patagonicus), rockhopper penguins (Eudyptes chrysocome), gentoo penguins (Pygoscelis papua), and Magellanic penguins (Spheniscus magellanicus). For all species except rockhopper penguins, primer pair 2550F/2718R resulted in two distinct CHD1Z and CHD1W PCR bands, allowing for sex identification. For rockhopper penguins, only primer pair p8/p2 yielded different CHD1Z and CHD1W bands, which were faint and similar in size making them difficult to distinguish. As a result, we designed a new primer pair (PL/PR) that efficiently determined the gender of individuals from all four penguin species. Sequencing of the PCR products confirmed that they were from the CHD1 gene region. Primer pair PL/PR can be evaluated for use in sexing other penguin species, which will be crucial for the management of new penguin breeding programs.

Characterization of the spotted seal Phoca largha transcriptome using Illumina paired-end sequencing and development of SSR markers.

Next-generation sequencing provides a powerful new approach for developing functional genomic tools for non-model species. This study aims to investigate the spotted seal (Phoca largha) transcriptome by the approach of Illumina paired-end sequencing technology. We obtained a total of 52,146,394 reads for the mixed tissues of liver and spleen from spotted seal. The de novo assemblies yielded 354,014 contigs and 178,466 unigenes. In addition, bioinformatics analysis revealed a total of 4425 simple sequence repeats (SSRs). Fifty SSRs were randomly selected to validate amplification and determine the degree of polymorphism in the genomic DNA pools. Thirty-five primer pairs successfully amplified expected DNA fragments and detected significant polymorphism among 28 spotted seal individuals. These results contribute to the understanding of the genetic makeup of spotted seal transcriptome and provide useful information for functional genomic research in this species.

Major histocompatibility complex class IIA and IIB genes of the spotted halibut Verasper variegatus: genomic structure, molecular polymorphism, and expression analysis.

The major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism and its association with resistance/susceptibility to disease in vertebrates. In this study, the full lengths of MHC IIA and IIB cDNA were obtained from spotted halibut (Verasper variegates) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The genomic structure, molecular polymorphism, and expression patterns were examined to study MHC II gene functions in fish. As in other teleosts, the genomic structure of the spotted halibut MHC IIA contained 4 exons and 3 introns. The deduced amino acid sequence of the class IIA molecule shared 28-79% similarity with those of teleosts and mammals. Nine class IIA alleles were identified from five individuals. Three alleles originating from a single individual suggested the existence of at least two class IIA loci in the genome. Six exons and 5 introns were identified from spotted halibut MHC IIB, and the deduced amino acid sequence shared 33-79% similarity with those of teleosts and mammals. Twelve alleles were identified, among which five were observed in a single individual, which suggested at least three class IIB loci. Quantitative real-time PCR analysis revealed the presence of class IIA and IIB transcripts in nine normal tissues with high expression level in kidney and gill. Furthermore, MHC IIA and IIB are probably two candidates of immune molecules involved in the acute-phase response in spotted halibut, because their transcriptional levels were significantly up-regulated in blood and liver after bacterial challenge.

Sequence and organization of the complete mitochondrial genomes of spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri).

In this work, the mitochondrial genomes for spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri) were completely sequenced. The entire mitochondrial genome sequences of the spotted halibut and barfin flounder were 17,273 and 17,588 bp in length, respectively. The organization of the two mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (2 rRNAs, 22 tRNAs and 13 protein-coding genes) and two non-coding regions (control region (CR) and WANCY region). In the CR, the termination associated sequence (ETAS), six central conserved block (CSB-A,B,C,D,E,F), three conserved sequence blocks (CSB1-3) and a region of 61-bp tandem repeat cluster at the end of CSB-3 were identified by similarity comparison with fishes and other vertebrates. The tandem repeat sequences show polymorphism among the different individuals of the two species. The complete mitochondrial genomes of spotted halibut and barfin flounder should be useful for evolutionary studies of flatfishes and other vertebrate species.

Echolocation click sounds from wild inshore finless porpoise (Neophocaena phocaenoides sunameri) with comparisons to the sonar of riverine N. p. asiaeorientalis.

Acoustic signals from wild Neophocaena phocaenoides sunameri were recorded in the waters off Liao-dong-wan Bay located in Bohai Sea, China. Signal analysis shows that N. p. sunameri produced "typical" phocoenid clicks. The peak frequencies f(p) of clicks ranged from 113 to 131 kHz with an average of 121+/-3.78 kHz (n=71). The 3 dB bandwidths delta f ranged from 10.9 to 25.0 kHz with an average of 17.5+/-3.30 kHz. The signal durations delta t ranged from 56 to 109 micros with an average 80+/-11.49 micros. The number of cycles N(c) ranged from 7 to 13 with an average of 9+/-1.48. With increasing peak frequency there was a faint tendency of decrease in bandwidth, which implies a nonconstant value of f(p)/delta f. On occasion there were some click trains with faint click energy presenting below 70 kHz, however, it was possibly introduced by interference effect from multiple pulses structures. The acoustic parameters of the clicks were compared between the investigated population and a riverine population of finless porpoise.